Giovanni Neri Istituto di Genetica Medica Università Cattolica del S. Cuore Roma

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1 Giovanni Neri Istituto di Genetica Medica Università Cattolica del S. Cuore Roma Fourth European Course on Clinical Dysmorphology Rome, November 25-26, 2011

2 A family of conditions that, although phenotypically different, are all related to the instability of a triplet repeat within the FMR1 gene, corresponding to the folate-sensistive fragile site FRAXA on chromosome Xq27.3: Fragile X syndrome(fxs) Fragile X tremor ataxia syndrome (FXTAS) Fragile X premature ovarian insufficiency (FXPOI)

3 THE MOST COMMON FORM OF HERITABLE INTELLECTUAL DISABILITY WITH A PREVALENCE OF 1:3,000, CORRESPONDING TO APPROXIMATELY ¼ OF ALL CASES OF XLID INHERITED AS A SEMIDOMINANT X-LINKED TRAIT, WITH HIGHER PENETRANCE AND EXPRESSIVITY IN MALES, COMPARED TO FEMALES

4 1943: The Martin-Bell syndrome 1969: Lubs discovery of a fragile site in the X chromosome of a XLID family 1981: The fragile site is demonstrated in the original Martin-Bell family 1991: Discovery of the FMR1 gene 1994: The mouse model of FXS 200..: The start of clinical trials

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7 KLC

8 From: Bagni and Greenough

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10 Macrocrania Elongated face with tall forehead and prominent jaw Malar hypoplasia Large ears Highly arched palate Macro-orchidism Joint hyperlaxity Mitral valve prolapse Congenital muscular hypotonia Brain MRI: increased size of caudate and hippocampus, cerebellar vermis hypoplasia Increase of dendritic spines

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17 ID of moderate degree ADHD Anxiety Impulsivity Perseveration of speech Avoidance of eye contact Hand flapping and other motoric stereotypies Autism Epilepsy (complex partial seizures) in 20% of cases

18 Southern blot continues to be the goldstandard

19 UPPER PANEL: MALE WITH A FULL MUTATION LOWER PANEL: MALE WITH A PREMUTATION

20 Which strategies for the pharmacological treatment of the fragile X syndrome?

21 Causal homogeneity Phenotypic consistency Apparent lack of irreversible damages Reversibility of the neuronal (dendritic) phenotype Some knowledge of the underlying molecular mechanisms Preservation of the ORF of the FMR1 gene

22 POSSIBLE THERAPEUTIC OPTIONS Gene therapy: currently not an option Stem cell therapy: currently not an option Pharmacological treatment targeted at: a. mglur receptors b. GABA receptors c. AMPA receptors d. NMDA receptors e. Dopaminergic and serotoninergic neurotransmission f. Metalloproteinase activity Drug-induced gene reactivation

23 FMRP absence results in excessive LTD and spine elongation/immaturity From Pirozzi et al, 2011

24 Multiple phamacological targets From Levenga et al, 2010

25 THE AFQ056 TRIAL AIM OF THE STUDY: To test whether therapeutic blockade of mglur5 by AFQ056 can improve behavioural symptoms in patients with FXS DESIGN OF THE STUDY: Double-blind, placebo-controlled, twotreatment, two-period crossover study DURATION OF THE TREATMENT: 28 days PATIENTS: 25 FXS men, aged years ENDPOINTS: ABC-C score (primary), CGI-I scale, CGI efficacy index

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27 The response of individual patients to AFQ056 and placebo treatment on the ABC-C at day 29-30, grouped by methylation status of the FMR1 promoter

28 28 SOS Ras/Rap1 MEKK3 MEK5 BMK1/ERK5 Raf MEK1/2 ERK1/2 Rac/Cdc42 PAK MEKK1 MKK4/MKK7 JNK TYK2 TRAF2 ASK1/2 MKK3/MKK6 P38 MAPK2 PKA AKT Myc AP1 MKP - - PI3K - PTEN TSC1/TSC2 PDK1 Rheb.GTP/mTOR/raptor S6K1 FMRP mglur5 Cell Cycle APP HRAS/KRAS/ Neurofibromin SHP2 MSK1 CREB

29 29 SOS Ras/Rap1 MEKK3 MEK5 BMK1/ERK5 Rac/Cdc42 MEKK1 MKK4/MKK7 JNK TYK2 TRAF2 ASK1/2 MKK3/MKK6 P38 MAPK2 PKA AKT Myc AP1 MKP - - PI3K - PTEN PDK1 Raf MEK1/2 ERK1/2 PAK TSC1/TSC2 Rheb.GTP/mTOR/raptor S6K1 FMRP mglur5 APP Cell Cycle HRAS/KRAS/ Neurofibromin SHP2 MSK1 CREB

30 PI3K Ras/Rap1 AKT PAK Raf HRAS/KRAS/ Neurofibromin MEK1/2 MKK3/MKK6 SHP2 mglur5 ERK1/2 MKP P38 MAPK2 MSK1 CREB TSC1/TSC2 Cell Cycle Rheb.GTP/mTOR/raptor S6K1 FMRP APP 30

31 POSSIBLE THERAPEUTIC APPROACHES Gene therapy: currently not an option Stem cell therapy: currently not an option Pharmacological treatment targetted at: a. mglur receptors b. GABA receptors c. AMPA receptors d. NMDA receptors e. Dopaminergic and serotoninergic neurotransmission f. Metalloproteinase activity Drug-induced gene reactivation

32 Epigenotype at the FMR1 promoter

33 In vitro reactivation of FMR1 Full Mutation DNA demethylation (5-azadC) Histone tail acetylation (HDAC inhibitors, ALC, VPA)

34 Our goal: convert a FM into an UFM

35 Epigenetic Modifications of FMR1 induced by 5-azadC Unmethylated K4 K9 CpG Methylated K4 K9 CpG Methylated full mutation (FXS) MBD MBD MBD MBD MBD MBD CpGs of the promoter methylated H3 and H4 deacetylated H3-K4 demethylated H3-K9 methylated 5-azadC Unmethylated full mutation (UFM) demethylation of CpGs of the promoter increase of H3 and H4 acetylation increase of H3-K4 methylation decrease of H3-K9 methylation CpGs of the promoter unmethylated H3 and H4 acetylated H3-K4 methylated H3K9 methylated Normal

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38 BOTH GROUPS (LAC AND PLACEBO) IMPROVED THEIR BEHAVIOUR, SHOWING THAT PSYCHOSOCIAL INTERVENTION HAS A SIGNIFICANT THERAPEUTIC EFFECT HOWEVER, REDUCTION OF HYPERACTIVITY WAS STRONGER IN THE LAC-TREATED GROUP, A SHOWN BY BOTH CONNERS GLOBAL INDEX AND VINELAND ADAPTIVE BEHAVIOUR SCALE THESE RESULTS SHOW THAT LAC REPRESENTS A SAFE ALTERNATIVE TO THE USE OF STIMULATING DRUGS FOR THE TREATMENT OF ADHD IN FXS BOYS

39 VPA treatments Tabolacci et al., Pharma Genomics, 2008 Maximum levels of FMR1 reactivation was around 3%, with an hyperacetylating effect.

40 AIM OF THE STUDY To perform an initial open label investigation of the effects of Sodium Valproate in children and adolescents with FXS SUBJECTS 10 children with FMR1 methylated full mutation DOSAGE Increasing dosage from 10 to 30 mg/kg/day for 6 months

41 PRIMARY ENDPOINT Effect of valproate on ADHD NEUROPSYCHOLOGICAL TESTS Conners Teacher Rating Scale - R (short form) Vineland Adaptive Behaviour Scale (VABS)-Survey Form Clinical Global Impressions (CGI) - Severity Teacher and Parent Rating Scale SNAP-IV Schedule for Affective Disorder and Schizofrenia for school age children (K-SADS)

42 CONNERS OPPOSITIONAL CONNERS COGNITIVE PROBLEMS NS 80 P = CONNERS HYPERACTIVITY CONNERS ADHD P = 0.03 P =

43 ALL MEASUREMENTS SHOWED AN IMPROVEMENT AFTER VPA TREATMENT IMPROVEMENTS MEASURED WITH THE CONNERS SCALE (HYPERACTIVITY) REACHED STATISTICAL SIGNIFICANCE

44 Rome Elisabetta Tabolacci Filomena Pirozzi Martina Goracci Marco Moscarda Maria Grazia Pomponi Stella Lanni Giorgia Mancano Sabrina Stefanini Roberta Pietrobono Collaborators Ben Oostra, Rotterdam, Erasmus University, Rotterdam Steve Haggarty, Broad Institute (Harvard-MIT), Cambridge Baltazar Gomez-Mancilla & Fabrizio Gasparini, Novartis, Basel Supported by: